Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol

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Abstract
Methanol oxidase isolated from Hansenula polymorpha contains 2 distinct flavin cofactors in approximately equal amounts. One has been identified as authentic FAD, and the other, as a modified form of FAD differing only in the ribityl portion of the ribityldiphosphoadenosine side chain. The significance of this finding is as yet unknown. Previous studies have shown that cyclopropanol irreversibly inactivates methanol oxidase [Mincey, T., Tayrien, G., Mildvan, A.S., and Abeles, R.H. (1980)]. Inactivation apparently is accompanied by covalent modification of the flavin cofactor. The stoichiometry of this reaction is 1 mol of cyclopropanol/mol of active flavin. The structure of the covalent adduct was determined by NMR, IR and UV spectra studies to be an N5, C4a-cyclic 4a,5-dihydroflavin. Reduction of the covalent adduct with NaBH4 at pH 9.0, before removal from the enzyme, converted it to the 1-(ribityldiphosphoadenosine)-substituted 4-(3-hydroxypropyl)-2,3-dioxoquinoxaline. Cyclopropyl ring cleavage accompanies inactivation, and covalent bond formation occurs between a methylene carbon of cyclopropanol and N5 of flavi. Methanol oxidase was also reconstituted with 5-deazaflavin adenine dinucleotide (dFAD). Reconstituted enzyme did not catalyze the oxidation of alcohols to the corresponding aldehydes, nor did reduced reconstituted enzyme catalyze the reverse reaction. Incubation of reconstituted enzyme with cyclopropanol resulted in an absorbance decrease at 399 nm, but not in an irreversible covalent modification of the deazaflavin cofactor. A reversible addition complex between cyclopropanol and dFAD is formed. The structure of that complex was not definitively established, but it is likely that it is formed through the addition of cyclopropoxide to C5 of dFAD. The failure of dFAD-reconstituted methanol oxidase to catalyze the oxidation of substrate, as well as the lack of reaction with cyclopropanol, supports a radical mechanism for alcohol oxidation and cyclopropanol inactivation. Methanol oxidase catalyzes the oxidation of cyclopropylcarbinol to the corresponding aldehyde. No ring-opened products were detected. The failure to form ring-opened products is used as an argument against radical processes [MacInnes, I., Nonhebel, D.C., Orsculik, S. T., and Suckling, C.J. (1982)]. Arguments against this interpretation are presented.

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